biology essay

Radiation Dose And The Radiation Quality Biology Essay

Published: 23, March 2015

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The biological effects of ionizing radiation are determined by both the radiation dose and the radiation quality ionization density. To understand the radiation protection concerns associated with different types of ionizing radiation, knowledge of both the extent of exposure and consequent macroscopic dose absorbed - gray value, as well as the microscopic dose distribution of the radiation modality is required. The definitions of these variables are discussed below but in general to advance the knowledge of the biological effects of different radiation types one needs to know the dose absorbed, the radiation quality and effectiveness of a particular radiation type to induce biological damage.

In this study the biological effect of high energy neutrons is compared to that of a reference radiation type 60Co Î³-rays for a cohort of donors, mostly radiation workers. Comparisons are made at different dose levels in blood cells from each donor to ascertain the relative biological effectiveness of the test radiation modality against that of a recognized reference radiation (Hall, 2005). Such studies are essential to determine the radiation quality for high energy neutron sources applicable to practises in radiation protection.

In some nuclear medicine applications radionuclides are used to treat malignant disease. For this the use of short lived alpha particle emitters or other radiation modalities that deliver high ionization densities in cells, are particularly attractive. This as the cellular response in relation to inherent radiosensitivity of the effected cells is thought to be more consistent compared to the use of radionuclides that emit radiation with a lower ionization density e.g. Î²-particles. The relative biological effectiveness of the high energy neutrons used in this study is followed as a function of the inherent radiosensitivity of different individuals. This allows the identification of cell populations that are relatively sensitive or relatively resistant to radiation. As such research material is available to investigate cellular response too Auger electrons. The latter is known to induce biological damage akin to that of alpha particles.

A short description of the physical and biological variables applicable to this study is summarised below.

Ionizing Radiation

The term ionizing radiation refers to both charged particles (e.g., electrons or protons) and uncharged particles (e.g., photons or neutrons) that can impart enough energy to atoms and molecules to cause ionizations in that medium, or to initiate nuclear or elementary-particle transformations that in turn result in ionization or the production of ionizing radiation. Ionization produced by particles is the process by which one or more electrons are liberated in collisions of the particles with atoms or molecules (The International Commission on Radiation Units and Measurements [ICRU] Report 85, 2011).

Interaction of Ionizing Radiation with Matter

Ionizing radiation is not restricted to ionization events alone. Several physical and chemical effects in matter such as: heat generation, atomic displacements, excitation of atoms and molecules, destruction of chemical bonds and nuclear reactions may occur. The effects of ionizing radiation on matter depend on the type and energy of radiation, the target, and the irradiation conditions.

Types of ionizing radiation linked to this study

Î³-rays

Ionizing photons (Î³- and X-rays) are indirectly ionising radiation. These wave like particles have zero rest mass and carry no electrical charge. Low energy (E<m0c2) photons only interact with orbital electrons and give rise to fast moving secondary electrons (Hall, 2005). The photons can be absorbed in photo-electric interactions or scattered and knock out atomic electrons in the process known as Compton scatter. If the energy (É¦Ê‹) of the incident photon exceeds 1.02 MeV the probability to create a pair of Î²- Î²+ particles in the pair production process dominates. Photons with very high energy (E>>2m0c2) may be absorbed by atomic nuclei and initiate nuclear reactions (Cember, 1969). The charged electrons emitted from the atoms, produce the excitation and ionisation events in the absorbing medium.

Neutrons

Neutrons, similar to ionizing photons are indirectly ionizing radiations; however, these particles do have a rest mass. There is negligible interaction between neutrons and the electron cloud of atoms since neutrons do not have a net electrical charge (Henry, 1969). The principle interactions occur through direct collisions with atomic nuclei during elastic scattering events. In this process, ionisation is produced by charged particles such as recoil nuclei and nuclear reaction products. The production of secondary ionising photons will result in the release of energetic electrons. In turn these charged particles can deposit energy at a considerable distance from the interaction sites (Pizzarello, 1982).

Auger electrons

Auger electron emission is an atomic-, not a nuclear process. In this process an electron is ejected from an orbital shell of the atom. A preceding event, e.g. electron capture (EC) or internal conversion (IC) leaves the atom with a vacant state in its electron configuration. An electron from a higher energy shell will drop into the vacant state and the energy difference will be emitted as a characteristic x-ray (Cember, 1969). The energy of the x-ray (Ex-ray) being the difference in energy (E) between the two electron shells L and K.

Ex-ray = EL -EK

Alternatively, the energy may be transferred to an electron of an outer shell, causing it to be ejected from the atom (Fig. 1). The emitted electron is known as an Auger electron and similarly to the x-ray has an energy:

Auger emission is favoured for, low-Z materials where electron binding energies are small. Auger electrons have low kinetic energies; hence travel only a very short range in the absorbing medium (Cember, 1969).

File:Auger Process.svg

Fig. 1: Schematic representation of the Auger electron emission process, where an orbital electron is ejected following an ionization event.

Dosimetric Quantities

Several dosimetric quantities have been defined to quantify energy deposition in a medium when ionizing radiation passes through it. Radiation fields are well described by physical quantities such as particle fluence or air kerma free in air are used. However these quantities do not relate to the effects of exposure on biological systems (International Commission on Radiological Protection [ICRP] Publication 103, 2007).

The absorbed dose, D, is the basic physical quantity used in radiobiology, radiology and radiation protection that quantifies energy deposition by any type of radiation in any absorbing material. The International System of Units (SI) of absorbed dose is joule per kilogram (J.kg-1) and is termed the gray (Gy).

Absorbed dose, D, is defined as the quotient of mean energy, dÎµ, imparted by ionising radiation in a volume element and the mass, dm, of the matter in that volume (Cember, 1969).

The absorbed dose quantifies the energy imparted per unit mass absorbing medium, but does not relate this value to radiation damage induced in cells or tissue.

The radiation weighted dose (HT) is used as a measure of the biological effect for a specific radiation quality on cells or tissue. It is calculated from equation

where DT,R is the mean absorbed dose in a tissue T due to radiation of type R and wR is the corresponding dimensionless radiation weighting factor. The unit of radiation weighted dose is J.kg-1 and is termed the sievert (Sv). Radiation weighting factors are recommended by the International Commission on Radiological Protection (International Commission on Radiological Protection [ICRP] Publication 103, 2007) and are derived from studies on the effect of the micro-deposition of radiation energy in tissue and on its carcinogenic potential.

Linear Energy Transfer (LET)

Ionizing radiation deposits energy in the form of ionizations along the track of the ionizing particle. The spatial distribution of these ionization events is related to the radiation type. The term linear energy transfer (LET) relates to the rate at which secondary charged particles deposit energy in the absorbing medium per unit distance (keV/Âµm). LET is a realistic measure of radiation quality (Duncan, 1977).

The LET (L) of charged particles in a medium is defined as the quotient of dE/dl where dE is the average energy locally imparted to the medium by a charged particle of specified energy in traversing a distance dl (Pizzarello, 1982).

For high energy photons (x- and Î³-rays), fast electrons are ejected when energetic photons interact with the absorbing medium. The primary ionization events along the track of the ionizing particle are well separated. This type of sparsely ionizing radiation is termed low-LET radiation. The LET of a 60Cobalt teletherapy source (1.3325 and 1.1732 MeV) is in the range of 0.24 keV/Âµm (Vral et al., 1994).

Neutrons cause the emission of recoil protons, alpha particles and heavy nuclear fragments during scattering events. These emitted charged particles interact more readily with the absorbing medium and cause densely spaced ionizing events along its track. The p66(Be) neutron beam used in this study has an ionization density of 20 keV/Âµm and hence regarded as high-LET radiation.

Auger electrons travel very short distances in the absorbing medium due to their low kinetic energies. All the energy of these particles is liberated in small volumes over short track lengths. Ionization densities are therefore very high, up to 40 keV/Âµm this is comparable to high-LET alpha particles (Godu et al., 1994).

Relative Biological Effectiveness (RBE)

The degree of damage caused by ionizing radiation depends firstly on the absorbed dose and secondly on the ionization density or quality of radiation. Variances in the biological effects of different radiation qualities can be described in terms of the relative biological effectiveness (RBE). RBE defines the magnitude of biological response for a certain radiation quality compared to a distinct reference radiation. It is expressed in terms of the ratio (Quoc, 2009):

Megavoltage X-rays or 60Co Î³-rays are commonly employed as the reference radiation since these are standard therapeutic sources of radiation.

Thus for an identical dose neutrons the biological effect observed would be greater, compared to 60Co Î³-rays. The fundamental difference between these radiation modalities is in the spatial orientation or micro deposition of energy. Furthermore, RBE varies as a function of the dose applied - increase in RBE is noted for a decrease in dose. By evaluating dose response curves (Fig 2), it is evident that the shoulder of the neutron curve is much shallower (smaller Î²-value) compared to the reference radiation curve. Therefore changes in RBE are prominent over low dose ranges (Hall, 2005).

Fig 2: Dose response curves based on the linear quadratic model demonstrate differences in RBE as a function of dose.

Through evaluation of the biological effect curves it is apparent that the RBE for a specific radiation quality may vary. This is characterized by the type of tissue or cells being investigated, dose and dose rates applied oxygenation status of the tissue, energy of radiation and the phase of the cell cycle and inherent radiosensitivity of cells. The RBE increases with a decrease in dose, to reach a maximum RBE denoted RBEM this is calculated from the ratio of the initial slope of the dose response curves for both radiation modalities.

RBE LET relationship

For a given absorbed dose, differences in the biological response for several cell lines, exposed to different radiation qualities have been demonstrated (Slabbert et al., 1996). Cells exposed to a specified dose low LET radiation do not exhibit the same biological endpoint than those exposed to same dose high LET radiation. This since with low LET radiation a substantial amount of damage may be repaired because the energy density imparted to each ionization site is relatively low. The predominant mode of interaction for this radiation type is indirect through chemical attack from radiolysis of water.

As the LET increases, for a specific dose, fewer sites are damaged but the sites that are located along the track of the ionizing particle are severely damaged because more energy is imparted. Thus the probability of direct interaction between the particle track and the target molecule increases with an increase in LET.

The RBE of radiation can be correlated with the estimates of LET values. However, as the LET increases, exceeding 10keV/Âµm it is no longer possible to assign a single value for the RBE. Beyond this LET, the shape of the cell survival curve changes markedly in the shoulder region compared to low-LET. Since RBE is a measure of the biological effect produced, comparison of the low-LET and high-LET curves will reveal that RBE increases with decreasing dose (Hall, 2005).

The average separation in ionizing events at LET of about 100 keV/Î¼m is equal to the width of deoxyribonucleic acid (DNA) double strand molecule (Fig. 3). Further increase in LET results in decreased RBE since ionization events occur at smaller intervals than DNA molecule strand separation (Fig. 3) and this energy imparted does not contribute to DNA damage.

Fig 3: Average spatial distribution of ionizing events for different LET values in relation to the DNA double helix structure (Hall 2005).

Cellular Radiosensitivity

Tissue radiosensitivity models

In 1906 the radiobiologists Bergonie and Tribondeau established a rule for tissue radiosensitivity. They studied the relative radiosensitivities of cells and from this could predict which type of cells would be more radiosensitive (Hall, 2005). Bergonie and Tribondeau realized that cells were most sensitive to radiation when they are:

Rapidly dividing (high mitotic activity).

Cells with a long dividing future.

Cells of an unspecialised type.

Ancel and Vitemberger later adapted the "law" of Bergonie and Tribondeau. They established that radiation damage was governed by two factors:

the biological stress on the cell.

pre and post irradiation conditions to which the cell is exposed.

Cell division causes biological stress thus cells with a short doubling time express radiation damage at an earlier stage than slowly dividing cells.

A comprehensive system of classification was proposed by Rubin and Casarett, cell populations were grouped into 4 categories based on the reproduction kinetics:

Vegetative intermitotic cells were defined as rapidly dividing undifferentiated cells. These cells usually have a short life cycle. For example: erythroblasts and intestinal crypt cells and are very radiosensitive.

Differentiating intermitotic cells are characterized as actively dividing cells with some level of differentiation. Examples include: meylocytes and midlevel cells in maturing cell lines these cells are radiosensitive.

Reverting postmitotic cells are regarded as to not divide regularly and generally long lived. Liver cells is an example of this cell type, these cell types exhibit a degree radioresistance.

Fixed postmitotic cells do not divide. Cells beloning to this classification are regarded to be highly differentiated and highly specialized in both morphology and function. These cells are replaced by differentiating cells in the cell maturation lines and are regarded as the most radioresistant cell types. Nerve and muscle cells are prime examples (Hall, 2005).

Thus according to the above classifications undifferentiated rapidly dividing cells are most radiosensitive (Hall, 2005).

Michalowski grouped tissues into hierarchical (H-type) and flexible (F-type) populations. His work, although novel is comparable to that of Rubin and Casarett.

Within the H- and F-type groupings, cells are assembled in 3 distinct categories:

Stem cells, continuously dividing cells that produce new stem cells and cells that eventually give rise to mature functional cells.

H-type populations have the capacity to divide unlimited. This population includes cell types such as the bone marrow, intestinal epithelium and epidermis.

F-type populations consist mainly of adult cells that will only divide under special conditions to produce another adult cell. This population includes cell types such as the liver parenchymal cells and thyroid cells.

H- and F-type groupings represent the extremes in cell populations. It should be noted that most tissue populations exist between the extremes. The lifespan and the reproductive potential of the primary cell type within that tissue is indicative of the radiosensitivity of that tissue (Hall, 2005).

Cell cycle dependent radiosensitivity

As cells progress through the cell cycle various physical and biochemical changes occur (Fig. 4). These changes influence the response of cells to ionizing radiation. Variations in radiosensitivity for several cell types at different stages of the cell cycle was previously noted (Hall, 2005). Following the law of Bergonie and Tribondeau that cells with high mitotic activity are most radiosensitive, it was found that cells in the mitotic phase (M-phase) of the cell cycle are indeed most sensitive. Late stage gap 2 (G2) phase cells are also very sensitive with gap 1 (G1) phase being more radioresistant and synthesis (S phase) cells the most resistant (Domon, 1980).

Fig. 4: Cell cycle of proliferating cells representing the different phases leading up to cell division. The G0 resting phase for cells that do not actively proliferate has been included since T-lymphocytes naturally occur in this phase (Hall, 2005).

Nonproliferating cells, generally cells that are fully differentiated, may enter the rest phase G0 from G1 and remain inactive for long periods of time. Peripheral T-lymphocytes seldom replicate naturally and remain in G0 indefinately.

Lymphocyte Radiosensitivity

The hematopoietic system is very sensitive to radiation. Differential blood analyses are routinely employed as a measure of radiation exposure. This measurement is based on the sensitivity of stem cells and the changes observed in the constituents of peripheral blood due to variations in transit time from stem cell to functioning cell (Hall, 2005).

It has been shown that lymphocytes, although they are resting cells (G0 phase) which do not actively proliferate nor do have a long dividing future hence do not meet the criteria of a radiosensitive cell type as described above are of the most radio sensitive cells. The reasons for their acute sensitivity cannot be explained (Hall, 2005).

Furthermore two distinct subpopulations T-lymphocytes with respect to radiosensitivity were found in peripheral blood. The small T-lymphocyte which is extremely radiosensitive and disappears almost completely from the peripheral blood at doses of 500 mGy (Kataoka, 1974, Knox, 1982 and Hall, 2005).

Cytogenetic expression of ionizing radiation induced damage

The primary target in radiotherapy is the double helix deoxyribonucleic acid (DNA) molecule (Rothkam et al. 2009). This macro molecule contains the genetic code critical to the development and functioning of most living organisms. The DNA molecule consists of two strands held together by hydrogen bonds between the bases. Each strand is made up of four types of nucleotides. A nucleotide consists of a five-carbon sugar (deoxyribose), a phosphate group and a nitrogen containing base. The nitrogen containing bases are adenine, guanine, thymine or cytosine. Base pairing between two nucleotide strands is universally constant with adenine pairing with thymine and guanine with cytosine (Fig. 5). This attribute permits effective single strand break repair since the opposite strand is used as a template during the repair process. The base sequence within a nucleotide strand differs; the arrangement of bases defines the genetic code. The double helix DNA molecule is wound up on histones and bound together by proteins to form nucleosomes. This structure is folded and coiled repeatedly to become a chromosome.

Fig. 5: The double helix structure of a DNA molecule consists of two neucleotide strands held together by hydrogen bonds between the bases. Figure modified from http://evolution.berkeley.edu/evolibrary/article/history_22 by P Beukes.

Ionizing radiation can either interact directly or indirectly with the DNA strand. When an ionization event occurs in close proximity to the DNA molecule direct ionization can denature the strand. Ionization events that occur within the medium surrounding the DNA produce free radicals such as hydrogen peroxide through radiolysis of water. Damage induced by ionizing radiation to the DNA include base damage (BD), single strand breaks (SSB), abasic sites (AS), DNA-protein cross-links (DPC), and double strand breaks (DSB) (Fig. 6).

Low-LET radiation primarily causes numerous single strand breaks, through direct and indirect interaction (Hall, 2005). Single strand breaks are of lesser biological importance since these are readily repaired by using the opposite strand as a template. High-LET radiation damage is dominated by direct interactions with the DNA molecule. Densely ionizing radiation has a greater probability to induce irreparable or lethal double strand breaks since energy deposition occurs in discrete tracks (Hall, 2005). The number of tracks will be fewer but more densely packed compared to low-LET radiation of equivalent doses.

Several techniques to quantify chromosomal damage and chromatid breaks have been established. These range from isolating DNA and passing it through a porous substrate or gel (Hall, 2005) by applying an external potential difference too advanced techniques of visually observing and numerating chromosomal aberrations of interphase cells.

Fig. 7: Different cytogenetic assays on peripheral T-lymphocytes for use in biological dosimetry. Figure modified from Cytogenetic Dosimetry IAEA, 2011.

PCC occurs when an interphase cell is fused with a mitotic cell. The fusion causes the interphase cell to produce condensed chromosomes prematurely. Chromosomal aberrations can thus be analysed immediately following irradiation without the need for mitogen stimulation or cell culturing.

Numeration of dicentrics in metaphase spreads has been used with great success to assess radiation damage in cells since the 1960's (Vral et al, 2010). The incidence of these aberrations follows a linear quadratic function with respect to the dose. Unstable aberrations like dicentrics or centric rings are lethal to the cell hence not passed on to daughter cells (Hall, 2005). In contrast translocations are stable aberrations; these are not lethal to the cell and passed on to daughter cells. Examination of translocations thus provides a long term history of exposure.

Although the abovementioned techniques are very accurate and well described, the complexity and time consuming nature of the assays has stimulated the development of automated methods of measuring chromosomal damage.

Micronuclei (MN) formation in peripheral blood T-lymphocytes lends itself to automation, since the outcome of radiation insult is visually not too complex with limited variables. DNA damage incurred from ionizing radiation or chemical clastogens induce the formation of acentric chromosome fragments and to a small extent malsegregation of whole chromosomes. Acentric chromosome fragments and whole chromosomes that are unable to engage with the mitotic spindle lag behind at anaphase (Cytogenetic Dosimetry IAEA, 2011). Micronuclei originate from these acentric chromosome fragments or whole chromosomes which are excluded from the main nuclei during the metaphase/anaphase transition of mitosis. The lagging chromosome fragment or whole chromosome forms a small separate nucleus visible in the cytoplasm of the cell. Image recognition software can thus be employed to quantify radiation damage by applying classifiers that describe cell size, staining intensity, cell separation, aspect ratio and cell characteristics when numerating MN frequency in BN cells. The classifiers are fully customizable depending on cell size, staining technique or cell type that will be used.

Rationale for this study

The principal objective of this study is to define RBE variations for high-LET radiation with respect to radiosensitivity. Specifically this is done for very high energy neutrons and Auger electrons. In general the response of different cell types vary much more to treatment with low-LET radiation compared to high-LET radiation (Broerse et al. 1978). Radiosensitivity differences have been demonstrated for different cancer cell lines (Slabbert et al. 1996) as well as various clonogenic mammalian cells (Hall, 2005) exposed to both high and low-LET radiation. In general there is an expectation and in certain cases some experimental evidence to support less variations in radiosensitivities of cells to high-LET radiation. Furthermore the ranking in the relative radiosensitivity of cell types changed for neutron treatments compared to exposure to X-rays (Broerse et al. 1978).

To quantify the radiation risk of individuals exposed to cosmic rays or mixed radiation fields of neutrons and Î³-rays, several experiments were conducted to ascertain biological damage induced by neutron beams of various energies (Nolte et al., 2007). Clonogenic survival data (Hall, 2005), dicentric chromosome aberrations (Heimers 1994) and micronuclei formation (Slabbert et. al 2010) have been followed. Chromosome aberration frequencies have been quantified and this represent radiation risk to neutron energies ranging from 36 keV up to 14.6 MeV (Schmid et al. 2003). To complement these studies additional measurements have been made for blood cells exposed to 60 MeV and 192 MeV quasi monoenergetic neutron beams (Nolte et al. 2007). Comparisons of RBE values obtained in these studies are shown in figure 1. Significant changes in the maximum relative biological effectiveness (RBEM) of these neutron sources are demonstrated as a function of neutron energy, with a maximum value of 90 at 0.4 MeV. RBEM drop to Â±15 for neutron energies higher than 10 MeV and it appears that the RBEM remain constant up to 200 MeV. The RBEM value of 47 -113 reported by Heimers et al. (1999) is not consistent with these observations.

The data shown in Fig. 1 was obtained by using the blood of a single donor. This to ensure consistency in the biological response for different neutron energies used in different radiation facilities in different parts of the world. Keeping the donor constant has the advantage that only a single data set for the reference radiation was needed. These measurements were done over several years. In all these studies, dicentric chromosome aberrations were followed. As informative as these investigations may be, it is doubtful if RBE values obtained from blood samples from a single donor are indeed representative for the wider population to state radiation weighting factors.

It is unclear if RBE values for high energy neutrons will vary when measured with cells with different inherent radiosensitivities. Warenius et al. (1994) demonstrated that the RBE of a 62.5 MeV neutron beam increases with increase in radioresistance to 6 MV X-rays. Similarly Slabbert et al (1996) using a 29 MeV p(66)/Be neutron with an average energy of 29 MeV, noted a statistically significant increase in the RBE values for cell types with increased radioresistance to 60Co Î³-rays. Although these investigators used 11 different cell types, few of these were indeed radioresistant to 60Co Î³-rays. Close inspection of the data shows that the relationship between neutron RBE and radioresistance to photons disappear when the cell type with the highest resistance to Î³-rays (Gurney melanoma) is removed from the data set Slabbert et al. 1996).

In a follow up study the authors failed to demonstrate the relationship for a p(66)/Be neutron beam but such a relationship was demonstrated for a d14/Be neutron beam (Slabbert et al. 2000). It therefore appears that the relationship for RBE and radioresistance is dependent on the selection of cells used in the study as well as the neutron energy.

Using lymphocytes Vral et al. (1994) demonstrated a clear reduction RBEM values for 5,5 MeV neutrons with an increase in the Î±-values of dose effect curves obtained for 60Co Î³-rays. This for lymphocytes obtained from six healthy donors. Using only four donors Slabbert et al. (2010) also demonstrated a relationship between RBEM neutrons and radiosensitivity to 60Co y rays. In the latter case the RBEM values are lower - as can be expected since these investigators used a higher energy neutron source. Although a significant relationship between these parameters has been demonstrated by the investigators, the cohort of 4 donors in the study is very small. In fact 2 out of the 4 donors have different RBEM values but appear to have the same radiosensitivity.

A study using larger number of donors with blood cells exposed to high energy neutrons is clearly needed. This in particular too verify the findings above indicating a different wR for donors of different sensitivity.

The studies of RBE variations with neutron energy by Schmid et al., (2003), Nolte et al. (2005) and were conducted dicentric formations observed in metaphase spreads. It is known that more than six months were used to analyse the data for different doses for blood cells obtained from a single donor exposed to a single neutron energy. It follows that some method of automation to assist the radiobiological evaluation of cellular radiation damage is needed to quantify wR values as a function of radiosensitivity.

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